Lens transparency is made possible by a combination of the highly ordered organization of the lens fiber cells, their unique refractive index, and the lack of organelles in the fiber cells. The highly ordered arrangement of the lens fiber cells is critical for proper light transmission, and disruption of this structure by alterations of cell-cell interactions is likely to lead to cataracts. However, signals that regulate lens fiber cell interaction remain largely unknown. Our preliminary studies have identified a new class of molecules, the Eph tyrosine kinase receptor family that regulates lens cell organization. Inactivation of ephrin-A5, a ligand of the Eph receptors, leads to the disruption of N-cadherin localization, change in lens fiber cell shape, disorganization of lens cells, and the development of cataracts. We hypothesize that ephrin-A5, interacting with its receptor(s), regulates N-cadherin-mediated fiber cell adhesion to maintain proper lens cell organization. To test this hypothesis, we will: (1) Examine the spatial and temporal characteristics of the ephrin-A5-null lens, determine when and where during development the lens defects first occur, and whether the loss of ephrin-A5 results in disruptions of lens fiber cell differentiation. The morphology of the lens at different developmental stages will be analyzed using both light and electron microscope techniques. Antibodies against markers of lens cell differentiation will be used in immunohistochemical experiments to study the expression of differentiation markers. (2) Elucidate receptor mechanisms of ephrin-A5 in lens development by examining which specific Eph receptors are expressed in the developing lens and where they are expressed, using Real-Time PCR, in situ hybridization, and immunohistochemistry. Since the interaction between Eph receptors and ligands leads to bidirectional signaling, we plan to analyze whether receptor- mediated signaling, the ligand-mediated reverse signaling or both are required for lens development using selective inactivation of different receptor domains. (3) Study the molecular alterations that lead to cataracts in ephrin-A5-null mice. Preliminary studies have revealed a disruption of N-cadherin distribution in the lens fiber cells. We will determine whether ephrin-A5 receptors interact physically with adherens junction molecules, and analyze effects of the ligand on N-cadherin functions. To critically evaluate roles of N-cadherin in mediating ephrin-A5 function and lens cell adhesion, we plan also to examine expression of a N-cadherin-2-catenin fusion protein in a phenotypic rescue experiment. The proposed studies will establish roles of a previously unsuspected family of molecules in lens development and reveal novel regulations of N-cadherin functions. These studies will enhance our understanding of how lens cell interaction is regulated to ensure lens transparency and provide insights into the mechanisms of cataractogenesis. PUBLIC HEALTH RELEVANCE: Cataracts are a leading cause of blindness. The molecular mechanisms underlie cataractogenesis are incompletely understood. The proposed studies will elucidate molecular mechanisms by which defects in ephrin-A5 signaling lead to cataracts and provide insights into future prevention and treatment of human cataracts.